The number of viral vectors designed for gene therapy applications in the cGMP pipeline is staggering. Similar in scope to the flurry of recombinant protein products of the 1980s and the monoclonal antibodies (MAbs) of the 1990s, viral vector-based products are surging from research labs and universities into contract manufacturing organizations (CMOs), ultimately destined for use in clinical trials. Unlike recombinant proteins and MAbs, both of which sometimes require grams of vialed final product to start Phase I studies, the amount of material required to move a viral vector-based product into clinical trials can be minute in comparison. Of all the viral vectors currently in clinical trials, more than 25% are based on adenovirus…
Tag: <span>adenovirus serotype 5</span>
The Adenovirus Reference Material Working Group (ARMWG) oversaw development of an adenovirus reference material (ARM) with the intent to provide a way to standardize assay measurements from different laboratories. The ARM, which was manufactured in stages by various organizations including Canji (San Diego, CA) and Introgen Therapeutics (Houston, TX), is available from American Type Culture Collection (Manassas, VA). Upon completion of its manufacture, the characterization phase primarily defined viral particle concentration as well as infectious titer for this product. However, many other concurrent characterization studies were conducted including an assessment of vector purity (e.g., host cell DNA, host cell protein, reversed-phase HPLC), a short-term field use and shipping stability study, and a long-term stability study. Also included in these studies was a coordinated effort to determine the complete DNA sequence of the ARM vector genome…
Recombinant adenovirus are attractive as vectors for gene therapy because: they exhibit wide tissue tropism and high transduction efficiency; adenovirus cultures can reach high specific titers (10^10 VP/mL), and; their use in the treatment of cancer and other serious diseases is valuable. A primary mode of adenovirus purification continues to be CsCl density gradient centrifugation…
In the past, researchers developing gene therapy applications used replication-defective human Adenovirus 5 (Ad5) as a vector for delivering DNA sequences, almost exclusively. Ad5 vectors are typically rendered replication defective by the deletion of E1a gene sequences. A complementing cell line containing the E1a gene makes it possible to produce Ad5 vectors in large scale. Of the various cell lines that have been constructed for the purpose of high-titer Ad5 production, HEK293 cells and PER.C6 cells are the most widely used…
The Adenovirus Reference Material (ARM) was developed under the guidance of the Adenovirus Reference Material Working Group (ARMWG) and the U.S. Food and Drug Administration (FDA), and was made possible through the donation of services and supplies by a large number of laboratories and institutions from the United States, Canada, France, The Netherlands, Germany, and the United Kingdom. The purpose of the ARM is to provide a reference material for use in validating assays and internal standards for adenoviral particle concentration and infectious titer. The NIH Recombinant DNA Advisory Committee recommended the development of such a reference-testing agent in their report issued January 2002. The ARM consists of purified wild type 5 Adenovirus as described by ATCC’s catalog number VR-5…
The Adenovirus Reference Material (ARM) is a purified and well-characterized wild type adenovirus (Ad5) now available to researchers worldwide. Due to the need for a common reference material, the ARM was produced with the purpose of validating assay methods and internal standards for use in developing recombinant adenovirus for gene therapy. Analysis of ARM by RP-HPLC, however, detected the presence of a contaminant peak with a distinctive A240 local wavelength maximum. The contaminant was found in all of the vials, with some variability in amount between vials. It appears that the contaminant is not associated with the virus and it is unlikely that it will interfere with the use of the ARM as a reference material. The source of the contaminant was probably a leachate or plasticizer from the tubing or containers used during the final processing step…
A Short-Term Field Use and Shipping Stability Study of a Wild Type Ad5 Adenoviral Reference Material
Adenoviral vectors for gene delivery are being tested in the clinic for a number of indications and therapeutic uses. In order to facilitate the comparison of studies from different laboratories, the Adenovirus Reference Material Working Group (ARMWG) has developed a reference testing reagent, which will be referred to as the Wild Type Ad5 Adenoviral Reference Material (ARM). This ARM will allow laboratories to standardize in-house controls employed in assays for the determination of particle concentration and infectious titer of their own adenoviral preparations. As part of this project, short-term field use and shipping studies were performed on the ARM. The virus was found to be stable under simulated shipping conditions, for one thaw after shipping, and at 4 °C for up to four hours after thawing. However, there was evidence of aggregation in some vials with repeated freeze-thaw cycles. Therefore, we recommend that each vial be treated as a single-use aliquot, and that it be used within four hours of thawing…